The long term objective of this project is to understand the detailed mechanisms controlling immunoglobulin heavy chain (IgH) expression, with emphasis on the principles of tissue-specific transcription regulation. One of the cis-acting transcriptional elements involved is a tissue- specific intronic enhancer. A plethora of different nuclear factors have been identified which bind to this element. One of these, the NF-muNR complex, plays an important role in the tissue-specificity of the enhancer, since it functions to repress enhancer activity in inappropriate cells. Two cDNAs have been isolated which apparently encode components of the NF-muNR complex since antibodies raised against each recombinant protein interacts with NF-muNR present in nuclear extracts. The specific aims of this proposal are to confirm the involvement of these two gene products and determine the complete multimeric structure using immunoprecipitation with specific antibodies, to determine if cell-type specific differences in expression, modification or compartmentalization occur for each, to analyze sequence requirements for DNA binding by isolating random oligonucleotides which preferentially bind each subunit separately and the holo-complex, to map these sites within the enhancer and evaluate their function in transfection studies, to determine the importance of these two genes in the development of a normal immune system through gene knockout approaches, to determine the structure/function relationship of domains in each protein in terms of protein-protein interaction, DNA binding and repressor function, and to investigate the functional interaction between NF-muNR and the nuclear matrix in transgenic mice. This analysis is relevant to several aspects of Ig gene regulation in that the proposed experiments address issues of cell-type specificity of IgH expression, expression regulation during B lineage differentiation, responses to external regulatory factors, and the relationship-between Ig gene expression and VDJ-type recombination. It also has important implications concerning general mechanisms of gene regulation. The observations described in this proposal are relatively novel in three regards: the idea that enhancer repression is important for tissue- specificity, the unusual nature of the components of this negative enhancer regulator and the potential role of the nuclear matrix in transcription regulation. These concepts together imply a mechanism for transcription regulation that is unprecedented, Analysis of the IgH enhancer is especially relevant to other health-related issues. A large body of evidence implicates this enhancer in tumorigenesis, in that a variety of cancers demonstrate specific chromosomal translocations placing the IgH enhancer next to a number of protooncogenes, resulting in oncogene activation through inappropriate expression. The investigation of an activity which can repress this enhancer has obvious therapeutic implications.